The goals of this proposal are to define the mechanisms by which alternatively spliced isoforms of the serine- threonine kinase, BRAF, are expressed in the context of melanoma targeted therapies and to elucidate how these isoforms mediate therapy resistance. Approximately 50% of melanoma patients harbor an activating mutation in BRAF, a protein in the extracellular-signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Recently approved therapies targeting this pathway have increased treatment options for melanoma patients but multiple resistance mechanisms inevitably arise leading to therapy failure. A fuller understanding of these mechanisms of resistance offers the possibility for improved treatment design and identification of additional therapeutic targets. One of the most prevalent resistant mechanisms is the aberrant splicing of BRAF, which has been identified in patients resistant to RAF inhibitor mono therapy, as well as in patients receiving combination RAF and MEK inhibitors. This alternatively spliced protein exhibits enhanced dimerization and downstream ERK1/2 signaling in the presence of RAF inhibitors. Through the successful completion of the following three aims, we will gain an understanding of the mechanisms underlying the expression of BRAF splice variants and the mechanisms by which BRAF splice variants confer resistance. In the first aim, we will elucidate the mechanism by which BRAF is alternatively spliced. RNA-seq analysis of in vivo derived BRAF splice variant expressing melanoma cells revealed altered expression of the SRRM family of splicing-related proteins. We hypothesize that the observed differences in expression of the SRRM protein family contributes to the prevalence of BRAF splice variants. The second aim will identify residues on the BRAF splice variants that are critical for continued ERK1/2 signaling in the presence of RAF inhibitor. All reported BRAF splice variants lack the regulatory Ras binding domain and a single 14-3-3 scaffolding protein binding site. We hypothesize that removal of the S365 14-3-3 binding site and retention of the S729 site promote kinase activity in the presence of RAF inhibitors. Finally, we aim to investigate the role that BRAF splicing plays in mediating resistance to next generation, paradox-breaking BRAF inhibitors. Current RAF inhibitors lead to the paradoxical activation of wild type BRAF in healthy cells. Plexxikon has recently developed a next-generation RAF inhibitor, PLX8394, which selectively targets mutant BRAF but does not elicit deleterious paradoxical responses. Studies have demonstrated the efficacy of PLX8394 in vitro; however, the resistance mechanisms against PLX8394 that will arise in vivo remain unstudied. We show that PLX8394 blocks BRAF splice variant dimerization in vitro and thus hypothesize that PLX8394 will block splice variant signaling in an in vivo xenograft model. The data generated in this proposal may implicate specific spliceosome components and protein signaling mechanisms as novel targets for treating patients with RAF inhibitor resistant melanoma.